Recently, in USA and Europe, studies have been conducted on the development of live vaccines using lactic acid bacteria, and on vehicles for delivering useful hormone drugs into the intestines, and on the establishment of efficient genetic resources therefor and the development of insertion vectors for lactic acid bacteria. Particularly, the utility of lactic acid bacteria as vaccine vehicles has been highly evaluated, because unmethylated CpG DNA, lipoteichoic acid, peptidoglycan and the like, which are contained in lactic acid bacteria in large amounts, are known to function as adjuvants. In addition, lactic acid bacteria have many advantages in that they can induce intestinal mucosal immunity, because they show resistance to bile acid and gastric acid to make it possible to deliver antigens to the intestines (Jos F. M. K. Seegers, Trends Biotechnol., 20:508, 2002).
However, in order for lactic acid bacteria to be used as vaccine vehicles, it is required to develop a technology of presenting antigen proteins for the production of disease-preventing antibodies to the inside or outside of bacterial cells so as to allow antigen-antibody reactions to occur smoothly. In fact, various study results, which indicate that lactic acid bacteria are suitable as vaccine vehicles, have been reported. Examples of these studies include the examination of the antibody-inducing capacity of lactic acid bacteria, in which the L1 protein of human papilloma virus (HPV) is expressed in inside (Karina, A. A. et al., Appl. Environ. Microbiol., 72: 745, 2006), and the examination of the disease-treating effects of a lactic acid bacterial strain which secrets and expresses IL-2 (interleukin-2) (Lothar, S. et al., Nat. Biotechnol., 21:785, 2003). As described above, the development of various applications of lactic acid bacteria expressing target proteins, and scientific studies on the lactic acid bacteria, have been actively conducted, but there are problems in that the expression levels of the target proteins are insufficient and expression vectors are unstable in host cells.
Methods for producing foreign proteins in host cells include: a method of using a highly efficient promoter to increase the expression level of the protein; a cell surface display method of expressing a desired protein by attaching it onto the surface of host cells; and a method of increasing the copy number of an expression vector in host cells.
The cell surface display technology uses surface proteins of microorganisms, such as bacteria or yeasts, as a surface anchoring motif, to express a foreign protein on the surface and is used in various applications, including production of recombinant live vaccines, construction and screening of peptide/antibody library, whole cell absorbents, whole cell bioconversion catalysts, and the like. The application scope of this technology is determined according to the kind of protein to be expressed on the cell surface. Therefore, it is considered that the cell surface display technology can be used in a very broad range of applications.
Previously, the present inventors conducted studies on the use of a poly-gamma-glutamic acid synthetase complex gene (pgsBCA), derived from Bacillus subtilis sp., as a novel surface anchoring motif, and as a result, developed a novel vector for effectively expressing a foreign protein on the surface of microorganisms and a method for expressing large amounts of a foreign protein on the surface of microorganisms, using the pgsBCA gene (Korean Patent Registration No. 469800).
In a method of using a highly efficient promoter, Known promoters for producing foreign proteins in lactic acid bacteria include constitutive expression promoters derived from the genome of Streptococcus thermophilus A504, Lactococcus lactis MG1614 or Lactococcus cremoris Wg2 (Philippe, S. et al., Appl. Envion. Microbial., 57:1333, 1991, Teija, K. et al., Appl. Envion. Microbial., 57:333, 1991, J. M. van der Vossen et al., Appl. Envion. Microbial., 53: 2452, 1987). Previously, the present inventors developed a constitutively high-expression vector containing an aldolase promoter derived from Lactobacillus casei (Korean Patent Laid-Open Publication No. 10-2008-0086161).
Also, studies on a method for increasing the copy number of an expression vector in a host cell have been conducted (Tomio, M. et al, Appl. Microbiol. Biotechnol. 28:170, 1988). In addition, studies on a method of changing the copy number of a plasmid in a cell using the RepE involved in the replication of the protein have also been conducted (Yashuo, K. et al., J. Bacteriology, 173:1064, 1991).
The RepE protein that is the replication initiator protein of the mini-F plasmid plays an important role in initiating replication from the origin, has a molecular weight of 29 kDa and binds to the 19-bp repeat sequence of ori2 (Maki, S. et al., Mol. Gen. Genet., 194:337, 1984; Tolun, A. et al., Mol. Gen. Genet. 186:372, 1982). Also, it has been reported that the RepE protein is involved in regulation of the copy number of the plasmid, and the frequency of initiation of replication in ori2 is determined by the concentration of the RepE protein in cell, whereby determining the copy number of the plasmid is determined (Tokino, T. et al., Proc. Natl. Acad. Sci. USA 83:4109, 1986).
Thus, efforts have been made to the copy number and stability of the plasmid by regulating the RepE protein. Also, there have been studies that the copy number of the plasmid was increased by point mutation of RepE (Kawasaki, Y. et el., J. Bacteriology, 173:1064, 1991), as well as studies that a RepE mutant protein resulting from a frame shift of the C-terminal region of the RepE protein acts as a repressor of transcription of a target protein (Matsunaga, F. et al., J. Bacteriology, 177:1994, 1995).
Accordingly, the present inventors have made extensive efforts to an expression vector, which is stable in transformed recombinant microorganisms and expresses a highly level of a target protein in the recombinant microorganisms, and as a result, have found that an expression vector, which contains a gene encoding a RepE protein containing a deletion of 21 amino acids in the C-terminal region of the RepE protein, stably expresses a high level of a target protein, thereby completing the present invention.